Human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application is divisional application of U.S. applicationSer. No. 10/116,326, filed on Apr. 4, 2002, now U.S. Pat. No. 6,777,545,issued 17th Aug. 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/282,036, which was filed on Apr. 6, 2001, both ofwhich are herein incorporated by reference in their entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosedpolynucleotides, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed polynucleotides, which can be used fordiagnosis, drug screening, clinical trial monitoring, the treatment ofdiseases and disorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate the phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given the physiological importance ofkinases, they have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode novel human proteins and thecorresponding amino acid sequences of these proteins. The novel humanproteins (NHPs), described for the first time herein, share structuralsimilarity with animal kinases, including, but not limited to,serine-threonine kinases, and carbon catabolite depressing kinases.Accordingly, the described NHPs encode novel kinases having homologuesand orthologs across a range of phyla and species.

The novel human polynucleotides described herein encode open readingframes (ORFs) encoding proteins of 778, 762, and 703 amino acids inlength (see respectively SEQ ID NOS:2, 4, and 6).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHPs, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof at least one of the described NHPs. When the unique NHP sequencesdescribed in SEQ ID NOS:1-6 are “knocked-out” they provide a method ofidentifying phenotypic expression of the particular gene, as well as amethod of assigning function to previously unknown genes. In addition,animals in which the unique NHP sequences described in SEQ ID NOS:1-6are “knocked-out” provide a unique source in which to elicit antibodiesto homologous and orthologous proteins, which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses. To these ends, gene trappedknockout ES cells have been generated in murine homologs of thedescribed NHPs.

Additionally, the unique NHP sequences described in SEQ ID NOS:1-6 areuseful for the identification of protein coding sequences, and mapping aunique gene to a particular chromosome. These sequences identifybiologically verified exon splice junctions, as opposed to splicejunctions that may have been bioinformatically predicted from genomicsequence alone. The sequences of the present invention are also usefulas additional DNA markers for restriction fragment length polymorphism(RFLP) analysis, and in forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists of, NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHPs and/or NHP products, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of the novel human ORFsencoding the described novel human kinase proteins.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHPs described for the first time herein are novel proteins that areexpressed in, inter alia, human cell lines and human brain, pituitary,cerebellum, spinal cord, lymph node, testis, adrenal gland, uterus,mammary gland, rectum, hypothalamus, ovary, fetal kidney, fetal lung,aorta, 6-, 9-, and 12-week old embryos, adenocarcinoma, osteosarcoma,embryonic carcinoma, and normal umbilical vein cells. The describedsequences were compiled from sequences available in GENBANK, and cDNAsgenerated from human adult and fetal brain, pituitary, hypothalamus,testis, and fetus mRNAs (Edge Biosystems, Gaithersburg, Md.) that wereidentified using primers generated from human genomic DNA.

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described polynucleotides, including the specificallydescribed NHPs, and the NHP products; (b) nucleotides that encode one ormore portions of a NHP that correspond to any functional domain(s), andthe polypeptide products specified by such nucleotide sequences,including, but not limited to, the novel regions of any activedomain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHPs, in which allor a part of at least one domain is deleted or altered, and thepolypeptide products specified by such nucleotide sequences, including,but not limited to, soluble proteins and peptides; (d) nucleotides thatencode chimeric fusion proteins containing all or a portion of a codingregion of a NHP, or one of its domains (e.g., a receptor/ligand bindingdomain, accessory protein/self-association domain, etc.) fused toanother peptide or polypeptide; or (e) therapeutic or diagnosticderivatives of the described polynucleotides, such as oligonucleotides,antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructs,comprising a sequence first disclosed in the Sequence Listing.

As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species, and mutant NHPs, whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, directedevolution as described in, for example, U.S. Pat. Nos. 5,837,458 or5,723,323, both of which are herein incorporated by reference). Theinvention also includes degenerate nucleic acid variants of thedisclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NOS:1, 3 or 5 (as measured by BLAST sequencecomparison analysis using, for example, the GCG sequence analysispackage, as described herein, using default parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP-encoding polynucleotides. Such hybridization conditionscan be highly stringent or less highly stringent, as described herein.In instances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80 bases long, or about 34 to about 45 baseslong, or any variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a microarray or high-throughput “chip” format).Additionally, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used to represent all or a portion of thedescribed NHP sequences. An oligonucleotide or polynucleotide sequencefirst disclosed in at least a portion of one or more of the sequences ofSEQ ID NOS:1-6 can be used as a hybridization probe, in conjunction witha solid support matrix/substrate (resins, beads, membranes, plastics,polymers, metal or metallized substrates, crystalline or polycrystallinesubstrates, etc.). Of particular note are spatially addressable arrays(i.e., gene chips, microtiter plates, etc.) of oligonucleotides andpolynucleotides, or corresponding oligopeptides and polypeptides,wherein at least one of the biopolymers present on the spatiallyaddressable array comprises an oligonucleotide or polynucleotidesequence first disclosed in at least one of the sequences of SEQ IDNOS:1-6, or an amino acid sequence encoded thereby. Methods forattaching biopolymers to, or synthesizing biopolymers on, solid supportmatrices, and conducting binding studies thereon, are disclosed in,inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305, 4,631,211,5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405, thedisclosures of which are herein incorporated by reference in theirentirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-6 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. Preferably the probes consist of 60nucleotides, and more preferably 25 nucleotides, from the sequencesfirst disclosed in SEQ ID NOS:1-6.

For example, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, the described polynucleotide sequences shalltypically comprise at least about two or three distinct oligonucleotidesequences of at least about 8 nucleotides in length that are each firstdisclosed in the described Sequence Listing. Such oligonucleotidesequences can begin at any nucleotide present within a sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions, andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-6 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-6 canalso be used in the identification, selection, and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets, and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the intended target of the drug. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ IDNOS:1-6 can be utilized in microarrays, or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-6 in silico, andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-6 can be used toidentify mutations associated with a particular disease, and also indiagnostic or prognostic assays.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence, inconjunction with the presence of one or more specific oligonucleotidesequence(s) first disclosed in SEQ ID NOS:1-6. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHP antisense molecules, useful, for example, in NHP generegulation and/or as antisense primers in amplification reactions of NHPnucleic acid sequences. With respect to NHP gene regulation, suchtechniques can be used to regulate biological functions. Further, suchsequences can be used as part of ribozyme and/or triple helix sequencesthat are also useful for NHP gene regulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupincluding, but not limited to, a phosphorothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

Low stringency conditions are well-known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (and periodic updates thereof), and Ausubel et al.,1989, supra.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, genomic DNA and/or cDNA obtained byreverse transcription of mRNA prepared from, for example, human ornon-human cell lines or tissue known to express, or suspected ofexpressing, an allele of a NHP gene.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known to express, orsuspected of expressing, a NHP gene). A reverse transcription (RT)reaction can be performed on the RNA using an oligonucleotide primerspecific for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” using a standard terminal transferase reaction, the hybrid maybe digested with RNase H, and second strand synthesis may then be primedwith a complementary primer. Thus, cDNA sequences upstream of theamplified fragment can be isolated. For a review of cloning strategiesthat can be used, see, e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown to express, or suspected of expressing, a NHP, in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well-known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPgene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of carrying, or known to carry, a mutantNHP allele (e.g., a person manifesting a NHP-associated phenotype suchas, for example, behavioral disorders, immune disorders, obesity, highblood pressure, etc.), or a cDNA library can be constructed using RNAfrom a tissue known to express, or suspected of expressing, a mutant NHPallele. A normal NHP gene, or any suitable fragment thereof, can then belabeled and used as a probe to identify the corresponding mutant NHPallele in such libraries. Clones containing mutant NHP sequences canthen be purified and subjected to sequence analysis according to methodswell-known to those skilled in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.).

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toa NHP are likely to cross-react with a corresponding mutant NHPexpression product. Library clones detected via their reaction with suchlabeled antibodies can be purified and subjected to sequence analysisaccording to methods well-known in the art.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos.5,830,721, 5,837,458, 6,117,679, and 5,723,323, which are hereinincorporated by reference in their entirety.

The invention also encompasses: (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators, and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

Where, as in the present instance, some of the described NHP peptides orpolypeptides are thought to be cytoplasmic or nuclear proteins (althoughprocessed forms or fragments can be secreted or membrane associated),expression systems can be engineered that produce soluble derivatives ofa NHP (corresponding to a NHP extracellular and/or intracellulardomains, or truncated polypeptides lacking one or more hydrophobicdomains) and/or NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP domain to an IgFc), NHP antibodies, andanti-idiotypic antibodies (including Fab fragments) that can be used intherapeutic applications. Preferably, the above expression systems areengineered to allow the desired peptide or polypeptide to be recoveredfrom the culture media.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHP,as well as compounds or nucleotide constructs that inhibit expression ofa NHP sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs, or inappropriately expressed NHPs, for the diagnosis ofdisease. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to NHPs,NHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists oragonists (including compounds that modulate or act on downstream targetsin a NHP-mediated pathway) can be used to directly treat diseases ordisorders. For instance, the administration of an effective amount of asoluble NHP, a NHP-IgFc fusion protein, or an anti-idiotypic antibody(or its Fab) that mimics the NHP, could activate or effectivelyantagonize the endogenous NHP or a protein interactive therewith.Nucleotide constructs encoding such NHP products can be used togenetically engineer host cells to express such products in vivo; thesegenetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules, can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The NHP Sequences

The cDNA sequences (SEQ ID NOS:1, 3, and 5) and corresponding deducedamino acid sequences (SEQ ID NOS:2, 4, and 6, respectively) of thedescribed NHPs are presented in the Sequence Listing.

Expression analysis has provided evidence that the described NHPs can beexpressed in a moderate range of human tissues. In addition toserine-threonine kinases, the described NHPs also share significantsimilarity to a range of additional kinase families, including kinasesassociated with signal transduction, from a variety of phyla andspecies. The genomic locus encoding the described NHPs is apparentlyencoded on human chromosome 19 (see GENBANK accession no. AC020922). Assuch, the described sequences are useful, inter alia, for mapping thecoding regions of the human genome, and particularly chromosome 19.

Given the physiological importance of protein kinases, they have beensubject to intense scrutiny, as exemplified and discussed in U.S. Pat.Nos. 5,756,289 and 5,817,479, herein incorporated by reference in theirentirety, which describe uses and utilities that are applicable to thedescribed NHPs.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees, may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to, pronuclear microinjection(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus-mediatedgene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem cells(Thompson et al., 1989, Cell 56:313-321); electroporation of embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated genetransfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review ofsuch techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry a NHPtransgene in all their cells, as well as animals that carry a transgenein some, but not all their cells, i.e., mosaic animals or somatic celltransgenic animals. A transgene may be integrated as a single transgene,or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. Atransgene may also be selectively introduced into and activated in aparticular cell-type by following, for example, the teaching of Lasko etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell-type of interest, and will be apparent to thoseof skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particularcell-type, thus inactivating the endogenous NHP gene in only thatcell-type, by following, for example, the teaching of Gu et al., 1994,Science 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

Once transgenic animals have been generated, the expression of therecombinant NHP sequence may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

The present invention also provides for “knock-in” animals. Knock-inanimals are those in which a polynucleotide sequence (i.e., a gene or acDNA) that the animal does not naturally have in its genome is insertedin such a way that it is expressed. Examples include, but are notlimited to, a human gene or cDNA used to replace its murine ortholog inthe mouse, a murine cDNA used to replace the murine gene in the mouse,and a human gene or cDNA or murine cDNA that is tagged with a reporterconstruct used to replace the murine ortholog or gene in the mouse. Suchreplacements can occur at the locus of the murine ortholog or gene, orat another specific site. Such knock-in animals are useful for the invivo study, testing and validation of, intra alia, human drug targets,as well as for compounds that are directed at the same, and therapeuticproteins.

5.2 NHPS and NHP Polypeptides

NHPs, NHP polypeptides, NHP peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products related to a NHP, and asreagents in assays for screening for compounds that can be used aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and diseases. Given the similarityinformation and expression data, the described NHPs can be targeted (bydrugs, oligonucleotides, antibodies, etc.) in order to treat disease, orto therapeutically augment the efficacy of therapeutic agents.

The Sequence Listing discloses the amino acid sequences encoded by thedescribed NHP-encoding polynucleotides. The NHPs display initiatormethionines that are present in DNA sequence contexts consistent witheucaryotic translation initiation sites. The NHPs do not displayconsensus signal sequences, which indicates that they may be cytoplasmicor possibly nuclear proteins, although they may also be secreted ormembrane associated (given the presence of several hydrophobic domains).

The NHP amino acid sequences of the invention include the amino acidsequences presented in the Sequence Listing, as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described herein are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference), are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHPs encoded by the presently described nucleotide sequences, asjudged by any of a number of criteria, including, but not limited to,the ability to bind and modify a NHP substrate, or the ability to effectan identical or complementary downstream pathway, or a change incellular metabolism (e.g., proteolytic activity, ion flux,phosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described herein, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where the NHP peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHP peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of aNHP, but to assess biological activity, e.g., in certain drug screeningassays.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreignpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. A NHP coding sequence can be cloned individually into anon-essential region (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a NHP coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted sequence isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHP product in infected hosts (e.g.,see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHP nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHP gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHP coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, may be provided. Furthermore, the initiation codonshould be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for the desired processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theNHP sequences described herein can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the NHP product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the NHPproduct.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine,phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Anotherexemplary system allows for the ready purification of non-denaturedfusion proteins expressed in human cell lines (Janknecht et al., 1991,Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the sequenceof interest is subcloned into a vaccinia recombination plasmid such thatthe sequence's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect a NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHPs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching an appropriate signal sequence to a NHP would alsotransport a NHP to a desired location within the cell. Alternativelytargeting of a NHP or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes: A Practical Approach”, New, R.R.C., ed., OxfordUniversity Press, N.Y., and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures, which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of NHPs to a target site or desired organ, wherethey cross the cell membrane and/or the nucleus where the NHPs can exerttheir functional activity. This goal may be achieved by coupling of aNHP to a cytokine or other ligand that provides targeting specificity,and/or to a protein transducing domain (see generally U.S. ProvisionalPatent Application Ser. Nos. 60/111,701 and 60/056,713, both of whichare herein incorporated by reference, for examples of such transducingsequences), to facilitate passage across cellular membranes, and canoptionally be engineered to include nuclear localization signals.

Additionally contemplated are oligopeptides that are modeled on an aminoacid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components which naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 Antibodies to NHP Products

Antibodies that specifically recognize one or more epitopes of a NHP,epitopes of conserved variants of a NHP, or peptide fragments of a NHP,are also encompassed by the invention. Such antibodies include, but arenot limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of a NHP. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes for the evaluation of the effect of test compounds onexpression and/or activity of a NHP expression product. Additionally,such antibodies can be used in conjunction with gene therapy to, forexample, evaluate normal and/or engineered NHP-expressing cells prior totheir introduction into a patient. Such antibodies may additionally beused in methods for the inhibition of abnormal NHP activity. Thus, suchantibodies may be utilized as a part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with a NHP, a NHP peptide (e.g., one corresponding to afunctional domain of a NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of a NHP,or mutated variants of a NHP. Such host animals may include, but are notlimited to, pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including, but not limited to, Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, chitosan, surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, and potentially useful human adjuvants such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Alternatively, the immuneresponse could be enhanced by combination and/or coupling with moleculessuch as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,ovalbumin, cholera toxin, or fragments thereof. Polyclonal antibodiesare heterogeneous populations of antibody molecules derived from thesera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole. et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class, includingIgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridomasproducing the mAbs of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger. et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454), by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity, canbe used. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine mAb and a human immunoglobulinconstant region. Such technologies are described in U.S. Pat. Nos.6,114,598, 6,075,181 and 5,877,397 and their respective disclosures,which are herein incorporated by reference in their entirety. Alsoencompassed by the present invention is the use of fully humanizedmonoclonal antibodies, as described in U.S. Pat. No. 6,150,584 andrespective disclosures, which are herein incorporated by reference intheir entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: F(ab′)₂ fragments, which can be produced by pepsin digestionof an antibody molecule; and Fab fragments, which can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well-known tothose skilled in the art (see, e.g., Greenspan and Bona, 1993, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438). For example,antibodies that bind to a NHP domain and competitively inhibit thebinding of a NHP to its cognate receptor/ligand can be used to generateanti-idiotypes that “mimic” the NHP and, therefore, bind, activate, orneutralize a NHP, NHP receptor, or NHP ligand. Such anti-idiotypicantibodies or Fab fragments of such anti-idiotypes can be used intherapeutic regimens involving a NHP-mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen a NHP, and thusnever been tolerized to a NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1. An isolated protein comprising the amino acid sequence of SEQ IDNO:4.